Multivirus-Specific T Cells for Prevention and/or Treatment of Viral Infections in the Immunocompromised Host
Viral infections account for substantial morbidity and mortality in the immunocompromised host, for example patients who have received allogeneic stem cell transplantation (SCT). Infections caused by persistent herpesviruses such as Epstein-Barr virus (EBV), cytomegalovirus (CMV), and herpes simplex virus, as well as by respiratory viruses such as respiratory syncytial virus, parainfluenza, and influenza are well known, whereas the importance of infections caused by adenovirus (Adv), BK virus, and human herpesvirus-6 have more recently been appreciated (Kim et al., 2007; Myers et al., 2007; Kadakia et al., 1996; Comoli et al., 2006; de Pagter et al., 2008). Although pharmacological agents are standard therapy for some infections, they have substantial toxicities, generate resistant variants, and are frequently ineffective.
Immunotherapeutic strategies to restore virus-specific immunity offer an attractive treatment alternative. Donor-derived EBV-specific cytotoxic T lymphocytes (CTLs) have been prepared using EBV-transformed lymphoblastoid cell lines (EBVLCLs) as a source of antigen, and infusion of these lines prevented and treated EBV-driven B cell lymphoproliferative diseases (EBV-LPDs) after allogeneic hematopoietic SCT (HSCT) (Heslop et al., 1996; Comoli et al., 2007) (Heslop et al., 2010) with minimal toxicity (Cruz et al, 2010).
Similarly, adoptively-transferred donor-derived CMV-specific CTL generated using CMV peptides, lysate, or vector-transduced antigen-presenting cells (APCs) were able to reconstitute immune responses against this virus and protect patients against the development of CMV disease or late recurrences (Riddell et al., 1992; Walter et al., 1995; Einsele et al., 2002a; Einsele et al., 2002b; Peggs et al., 2003; Micklethwaite et al., 2008; Micklethwaite et al., 2007). More recently, trivirus-reactive CTL targeting EBV, CMV, and Adv were produced by genetically modifying monocytes and EBV-LCL with a chimeric adenoviral vector expressing CMV-pp65 as a transgene.
As few as 2×105/kg trivirus-specific CTL proliferated by several logs after infusion into HSCT recipients and appeared to protect the recipients against disease produced by all three viruses (Leen et al., 2006). Despite these encouraging clinical results, the broader implementation of T-cell immunotherapy is limited by (i) the infectious virus material (EBV/CMV/Adv) generally required for CTL generation, (ii) the expense of manufacture of clinical grade vectors for antigen presentation, and (iii) the prolonged period of culture (10-12-week manufacturing process) that is required to eliminate alloreactive T cells, with its attendant demands on technical skill and time. To address this latter problem, some groups have evaluated more rapid approaches for antigen-specific T-cell selection.
Cobbold and colleagues selected CMV-specific CD8+ T cells from the blood of stem cell transplant donors using human leukocyte antigen (HLA)-peptide tetramers followed by selection with magnetic beads, and saw impressive clinical responses with eight of nine treated patients clearing their infection following infusion of tiny numbers of selected cells (median 8.6×103/kg) (Cobbold et al., 2005). Selection of T cells that secrete interferon-γ (IFN-γ) after exposure to antigen (the IFN-γ capture assay) has also been used clinically by Feuchtinger and colleagues, who specifically selected Adv-specific T cells directly from peripheral blood after in vitro stimulation with viral peptides and showed that small numbers of cells (1.2-50×103/kg) were safe, protective, and effective in vivo (Feuchtinger et al., 2004; Feuchtinger et al., 2006). However, both these approaches are expensive and require a large starting blood volume, which is not always available, particularly in the matched unrelated donor setting. Tetramer reagents are limited already known epitopes and to CD8 selection. Gamma capture limits the infusions to T cells that secrete gamma interferon. Antigen-specific T cells with other functions may be lost So far, their use has been limited to restricted cases of urgent medical need.
The art needs an alternative good manufacturing practice-compliant method that overcomes all three limitations (volume, time, and infectious agents) to allow the rapid generation of multivirus-specific CTL from small blood volumes for immunotherapeutic purposes. The present invention provides how dendritic cells (DCs) can be nucleofected with DNA plasmids encoding a range of immunodominant and subdominant viral antigens and used as in vitro T-cell stimulators to generate multivirus-reactive CTLs that target multiple different epitopes in antigens from multiple common viruses within 10 days (Gerdemann et al., 2009).
Multi-Tumor Associated Antigen (TAA) T Cells for Prevention and/or Treatment of Cancer
For example, EBV is associated with Hodgkin's and non-Hodgkin's lymphoma and nasopharyngeal carcinoma. In these cases, the tumor cells express three of about 90 Epstein-Barr viral antigens. To optimize the antigenic targeting of CTLs, our group has prepared CTLs whose specificity was directed towards the three expressed viral antigens by sequentially using dendritic cells (DCs) and then EBV-LCL genetically modified to overexpress LMP1 and LMP2 (two of the three antigens) to reactivate and expand LMP-specific CTLs from patients or their HLA-matched allogeneic donors. The LMP antigens were expressed from an adenoviral vector. In HL and NHL the clinical results were encouraging, and 16 of 17 patients treated in remission of high-risk HL remained in remission, while 12/15 patients with active relapsed disease had tumor responses. However, >70% of the patients referred to our studies have EBV negative lymphomas and are not eligible for EBV antigen-specific T cells.
One of the challenges of adoptive immunotherapy for non-viral cancers remains the identification of strongly immunogenic target antigens. The model tumor antigens should be specifically and universally expressed on tumor cells in order to limit collateral damage, and ideally should be essential for the maintenance of the oncogenic phenotype of the tumor. However, the majority of antigens do not meet these criteria since they are not necessarily neo-antigens uniquely present in cancer cells but rather antigens that are also expressed in normal cells and against which peripheral blood T cells are tolerized or deleted. However antigens that are essentially tumor-specific have been identified, but the pattern of tumor antigens expressed is highly tumor type-dependent. This underscores the importance of identifying appropriate antigens that are not expressed or poorly expressed on normal tissues and of optimizing cell culture conditions for tumor-specific CTL production, to overcome the mechanisms that establish T cell tolerance against “self” antigens.
T cell immunotherapies for non-viral tumor antigen have been described, with promising clinical results in some studies. Rosenberg and colleagues reported that infusion of melanoma-specific tumor-infiltrating lymphocytes (TILs) together with high-dose interleukin 2 (IL-2) produced clinical responses in approx. 35% of patients with metastatic melanoma. The specificity of the infused cells was not analyzed but it is likely that they targeted multiple epitopes/tumor associated antigens. More promising results were subsequently achieved using a modified treatment protocol which incorporated a lymphodepletion step prior to CTL infusion, in order to improve the expansion and persistence of adoptively-transferred cells. Ninety three patients with metastatic melanoma refractory to standard therapies received immunodepleting chemotherapy −/+ total body irradiation followed by the adoptive transfer of highly selected, TIL-derived, tumor-reactive T cells and high-dose IL-2 (720,000 IU/kg q 8 h to tolerance). Fifty two of the 93 patients had objective clinical responses to treatment (39 PR, 12 CR), including regression of large bulky tumors. However, the collection of autologous TILs is not possible for most tumors. Furthermore the in vitro expansion of large numbers of tumor-specific T cells (>1010 CTL) is a complex and expensive procedure. The same group also infused T cell clones directed targeting a single epitope in the melanoma-associated antigen, gp100+/−IL-2 but reported poor clinical responses, with only one minor response and one mixed response, and showed that the cells failed to engraft or persist in vivo. These studies demonstrate the potential of adoptively-transferred antigen-specific T cells to eliminate cancer, but highlights the importance of targeting multiple epitopes/antigens to produce optimal clinical results.